This article was downloaded by: [University of Ottawa] On: 23 November 2014, At: 09:22 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
International Journal of Environmental Health Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cije20
Role of low exposure to metals as male reproductive toxicants a
b
c
Hueiwang Anna Jeng , Yeou-Lih Huang , Chih-Hong Pan & Norou d
Diawara a
School of Community and Environmental Health, Old Dominion University, Norfolk, VA, USA b
Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan c
Occupational Safety and Health, Occupational Medicine, Taipei, Taiwan d
Mathematics & Statistics Department, Old Dominion University, Norfolk, VA, USA Published online: 01 Oct 2014.
To cite this article: Hueiwang Anna Jeng, Yeou-Lih Huang, Chih-Hong Pan & Norou Diawara (2014): Role of low exposure to metals as male reproductive toxicants, International Journal of Environmental Health Research, DOI: 10.1080/09603123.2014.958137 To link to this article: http://dx.doi.org/10.1080/09603123.2014.958137
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
Downloaded by [University of Ottawa] at 09:22 23 November 2014
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
International Journal of Environmental Health Research, 2014 http://dx.doi.org/10.1080/09603123.2014.958137
Role of low exposure to metals as male reproductive toxicants Hueiwang Anna Jenga*, Yeou-Lih Huangb, Chih-Hong Panc and Norou Diawarad a
School of Community and Environmental Health, Old Dominion University, Norfolk, VA, USA; Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan; cOccupational Safety and Health, Occupational Medicine, Taipei, Taiwan; dMathematics & Statistics Department, Old Dominion University, Norfolk, VA, USA
Downloaded by [University of Ottawa] at 09:22 23 November 2014
b
(Received 31 March 2014; final version received 8 July 2014) The objective of the study was to examine the associations between environmentally relevant low metal concentrations and semen quality parameters in men. The concentrations of zinc (Zn), copper (Cu), cadmium (Cd), arsenic (As), selenium (Se), and lead (Pb) in the seminal plasma and urine were measured from 196 male human subjects in Taiwan. Urinary Cd concentrations were negatively associated with sperm viability (p = 0.006). Seminal plasma Cu concentrations of the normal group (≥ 15 × 106/ml) were significantly lower than those of the abnormal group (p = 0.023). However, the linear regression analysis showed a weak association between Cu concentration and sperm concentration, along with other semen parameters. No significant relationship between other metals (As, Pb, Zn, and Se) and semen quality was observed. Keywords: sperm concentration; viability; motility; morphology; metals
Introduction Due to the widespread presence of metals in the environment, the general population is exposed to low concentrations of metals through intake of contaminated food and water, contact with contaminated soil, or by breathing in dust or air (Meeker et al. 2008). Metals, such as lead (Pb), copper (Cu), zinc (Zn), and cadmium (Cd), have been reportedly linked with alteration of semen quality. Seminal plasma Pb of paint factory workers and the general population adversely affected sperm motility, morphology, and concentration (Eibensteiner et al. 2005; Naha & Manna 2007; Telisman et al. 2007; Meeker et al. 2008). Cd is also among the metals being found to associate with reduced sperm concentration (Telisman et al. 2000; Xu et al. 2003; Colagar & Marzony 2009), motility (Xu et al. 2003), and percentages of normal sperm morphology and viability (Xu et al. 2003; Colagar & Marzony 2009). Cu is necessary and useful for maintaining good reproductive health since it may act as a cofactor for a variety of important enzymes required for sperm development (Huang et al. 2000; Telisman et al. 2000). Despite its beneficial role, above certain levels, Cu proves to be harmful (Telisman et al. 2000). Similar to Cu, Zn could be beneficial for male reproductive health by acting as one of the factors responsible for antibacterial activity of the seminal plasma or possibly playing a role in sperm production, sperm viability, and degradation. Zn deficiency could lead to decreased testicular *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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H.A. Jeng et al.
weight, shrinkage of seminiferous tubules and gonadal dysfunction (Chia et al. 2000). There have been conflicting results about correlations of Zn concentrations in human seminal plasma and semen quality, including sperm concentration, motility, and viability (Dawson et al. 1998; Chia et al. 2000). To date, human data on some metals have been limited (e.g. As) or inconsistent across studies (e.g. Zn). Also, there is limited data on simultaneously examining semen quality associated with metals in different biological matrixes, such as urine and seminal plasma. Furthermore, there is limited data on the potential role of low, environmentally relevant exposure to metals in general populations in relation to male reproductive outcomes. This study aimed to examine the association between exposure to low concentrations of trace metals in seminal plasma and urine and semen quality (concentration, morphology, viability, and motility) of Taiwanese males in the general population. Semen quality serves as biological endpoints to assess the reproductive status of male individuals in this study and determine potential reproductive toxicity of metals. Materials and methods Human subjects A total of 281 human subjects were screened to determine eligibility for participation in this cross-sectional study. The screening occurred when they had their annual health examination at the Kaohsiung Municipal Hsiao-Kang Hospital, a main municipal hospital in the southern region of Taiwan. The criteria for selecting human subjects included males between 20 and 65 years old, no medical treatments affecting spermatogenesis (chemotherapy, radiotherapy, and vasectomy), no self-reported diabetes mellitus, cirrhosis or any other liver disease or neoplastic condition, no use of mood stabilizers, medications that affect the endocrine system, and no obesity class II and III according to the National Institutes of Health guidelines (NIH 2000). The status of fertility was not included as a criterion, since that reproductive status was not the focus of this study. However, we excluded those who had medical issues that prevented them from providing semen voluntarily. A total of 213 human subjects were found eligible and included in this study. The sample size was sufficient to yield a power of 85 % with a one-way analysis of variance at the 5 % level of significance. All participants were fully informed about the objective of this study and signed the consent form. The participants voluntarily provided semen and a spot of urine. They filled out a questionnaire describing demographic characteristics (age, height, weight, education, and marital status), smoking status, occupational exposure, and second-hand smoke exposure. Body mass index (BMI) for each human subject was calculated based on height and weight. The study was approved by the Institutional Review Boards at Old Dominion University and Kaohsiung Medical University. Semen quality analysis A semen sample was produced on-site by masturbation into a sterile plastic specimen cup. After collection, the sample was liquefied at 37 °C for 20 min before analysis. The participants were instructed to abstain from ejaculation for at least three days before semen collection. Each semen sample was collected by masturbation. All semen samples were analyzed for sperm concentration and motility according to World Health Organization guidelines (WHO 2010). Evaluation of percentage motility was done by manual
International Journal of Environmental Health Research
3
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assessment to quantify percentage of moving sperm compared with non-motile cells for at least 10 microscopic fields. For the morphology assessment, 300 sperm per sample were evaluated from air-dried Papanicolaou-stained preparations and classified as either normal or abnormal according to WHO’s strict criteria (WHO 2010). Results were expressed as percent normal sperm. Total sperm count was calculated as the product between sperm concentration and ejaculate volume. Total motile sperm was calculated as the product between total sperm count and progressive motility. All of the procedures were performed by the same laboratory technician. The mean inter-individual coefficients of variation were 18.2, 12.3, and 15.2 % for sperm concentration, motility, and viability, respectively Determination of metals in seminal plasma The semen samples were centrifuged at 1000 rpm for 5 min to separate sperm and seminal plasma. Then, 300 μL of each seminal plasma sample was transferred into a pre-cleaned 1.5 mL microtube and treated with 300 μL concentrated nitric acid at room temperature for 20 min. After homogenization, the mixture was diluted 5, 5, 1, 200, and 5-fold with deionized water for Cd, Cu, Pb, Zn, and As, respectively. After vigorous shaking, the sample solution was centrifuged at 12,000 rpm for 5 min. The obtained supernatant was directly analyzed by flame atomic absorption spectrometry (FAAS) for Zn, electrothermal atomic absorption spectrometry (ETAAS) for Cu, Cd, Se, and Pb, and hydride generation atomic absorption spectrometry (HGAAS) for As. The accuracy was validated by the standard spiked method, and the As, Cd, Cu, Pb, Se, and Zn concentrations in spiked semen samples were obtained by similar analytical methods. Recovery of the target analytes in spiked semen ranged from 95.10 to 105.97 %. The calibration curves were obtained after analyzing the standard series by FAAS, ETAAS, HGAAS as described, respectively. A good linearity was obtained at the concentration range of 5–30 (r2 = 0.9973), 0.5–2.5 (r2 = 0.9999), 5–50 (r2 = 0.9990), 5–60 (r2 = 0.9997), 1–6 (r2 = 0.9991), and 250–1000 (r2 = 0.9989) μg/L for As, Cd, Cu, Pb, Se, and Zn, respectively. Limits of detection (LOD) for As, Cd, Cu, Pb, Se, and Zn were 2.59, 0.03, 1.03, 0.98, 0.02, and 0.065 μg/L. Limits of quantitation (LOQ) for As, Cd, Cu, Pb, Se, and Zn were 8.46, 0.09, 2.95, 3.14, 0.07, and 0.217 μg/L, respectively. The concentrations of metals in seminal plasma were expressed as μg/L. Determination of metals in urine The urine samples were prepared as follows: 500 μL of urine was transferred into a precleaned 4 mL microtube and treated with aliquot of concentrated nitric acid at room temperature for 20 min. Then, the mixture solution was diluted 1, 1, 1, and 200-fold with deionized water for Cd, Cu, Pb, and Zn, respectively. After vigorous shaking, the urine samples were directly analyzed by FAAS for Zn, ETAAS for Cu, Cd, Pb, and Se. For As, urine samples were filtrated using a 0.45 μm hydrophilic polyethersulfone membrane prior to measurement by HGAAS. The accuracy was validated by determining the contents of As, Cd, Cu, Pb, Se, and Zn in standard reference material NIST-2670a human urine. The urine SRM 2670a was diluted similarly prior to analysis and the recoveries ranged from 93.05 to 108.17 %. The accuracy was also validated by the standard spiked method, with the As, Cd, Cu, Pb, Se, and Zn concentrations in spiked semen samples obtained by similar analytical methods. Recovery of the target analytes in spiked semen ranged from 93.05 to 104.91 %. The calibration curves were obtained at the
47.6 52.4 28.6 1.6 69.8 19.3 56.5 24.2
50.9 49.1
30.5 2.4 67.1
30.5 49.4 20.1
33.3 16.7 50.0
24.2 1.3 74.5
50.0 50.0
35.7 ± 11.6 24.2 ± 1.8
Concentration < 20 × 106/ml N=6
48.6 40.0 11.4
20.6 2.9 76.5
48.6 51.4
43.8 ± 9.4 25.6 ± 4.4
Motility < 40 % N = 31
61.9 23.8 14.3
23.8 4.8 71.4
50.0 50.0
46.1 ± 8.6 25.9 ± 4.1
Vitality < 58 % N = 21
45.5 39.4 15.2
36.9 3.1 60.0
60.6 39.4
38.7 ± 10.0 25.1 ± 4.4
Normal morphology < 4 % N = 66
*The comparison group was comprised of men with all four semen parameters higher than the reference levels according to the WHO reference levels.
36.8 ± 9.7 24.8 ± 3.9
Comparison group* N = 62
38.4 ± 9.9 25.0 ± 4.1
All
Demographic characteristics of the participants by semen quality parameters classified according to the WHO reference levels.
Age (years, mean ± SD) BMI (kg/m2, mean ± SD) Smoking status (%) Yes No Drinking (%) Yes Yes/Quit No Education (%) < High school High school College/Post college
Table 1.
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4 H.A. Jeng et al.
International Journal of Environmental Health Research
5
concentration range of 5–60 (r2 = 0.9976), 1–5 (r2 = 0.9998), 10–80 (r2 = 0.9991), 10–90 (r2 = 0.9994), 20–100 (r2 = 0.9991), and 100–800 (r2 = 0.9999) μg/L for As, Cd, Cu, Pb, Se, and Zn, respectively. The concentrations of As, Cd, Cu, Zn, Pb, and Se in urine samples were obtained by the general standard curve method. Concentrations of metals were adjusted by using urinary creatinine concentrations and expressed as ng/g creatinine. LOD for As, Cd, Cu, Pb, Se, and Zn were 2.59, 0.03, 1.03, 0.98, 0.02, and 0.065 μg/L, respectively. LOQ for As, Cd, Cu, Pb, Se, and Zn were 8.46, 0.09, 2.95, 3.14, 0.07, 0.217 μg/L, respectively. Urine volumes were adjusted by creatinine levels. The concentrations of metals were expressed as μg/g of creatinine.
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Statistical analysis Descriptive analysis was conducted to determine the mean distribution of demographics, semen quality parameters, and metal concentration in seminal plasma and urine. Mean concentration of each metal was calculated based on two separate groups of each semen parameter based on the WHO reference values. Mean metal concentrations in two categories of each semen parameter were compared using the Student’s t-test after ascertaining homogeneity of variance between the two groups. Association between exposure categories for each metal and sperm concentration, motility and vitality was assessed by binary logistic regression, where subjects were dichotomized in categories less than or greater Table 2. Distribution of semen quality and metals in seminal plasma, urine, and urine adjusted for creatinine levels. All Metal concentration Zn Cd Cu As Se Pb Metal concentration Zn Cd Cu As Se Pb Metal concentration Zn Cd Cu As Se Pb Semen quality Concentration (106/ml) Motility (% motile) Morphology (% normal) Viability (%)
10th
25th
50th
75th
in seminal plasma (μg/L) 169.3 ± 97.9 70.6 101.0 140.0 208.8 0.5 ± 0.3 0.38 0.4 0.6 1.4 170.8 ± 210.1 66.9 81.7 116.2 170.4 4.8 ± 5.5 1.3 1.3 1.3 7.3 249.1 ± 89.1 132.1 174.9 247.1 315.2 0.6 ± 1.1 0.5 0.5 0.5 0.5 in urine (μg/L) 602.0 ± 576.0 98.2 176.0 447.2 849.4 0.7 ± 0.6 0.2 0.4 0.9 1.4 14.8 ± 13.9 3.1 5.2 11.5 20.3 133.2 ± 146.4 12.7 33.3 77.1 177.6 20.4 ± 13.8 8.3 12.4 17.4 24.7 5.4 ± 6.4 0.3 0.5 3.1 8.5 in urine adjusted for creatinine (μg/g creatinine) 419.0 ±345.8 147.2 244.7 349.4 502.0 0.5 ± 0.4 0.1 0.2 0.4 0.8 12.3 ± 11.6 3.8 6.0 10.1 13.8 91.1 ± 91.9 19.4 37.4 64.1 110.8 15.4 ± 10.8 5.7 7.5 12.6 20.3 4.4 ± 7.8 0.2 0.8 2.5 6.1 111.5 190.0
90th
95th
Maximum
304.6 1.5 252.2 12.7 353.3 0.5
371.2 1.5 440.1 16.3 362.5 0.5
612.0 1.5 1756.8 32.8 609.7 12.6
1414.3 1669.3 2.4 5.8 31.8 36.1 336.1 433.8 35.6 45.7 15.1 17.8
3279.6 2.9 94.1 754.5 84.3 34.7
707.8 1.1 22.1 184.5 31.0 8.5
819.7 1.4 32.5 297.7 39.3 12.1
2938 2 88 686 58 72
273.0
313.5
905.0
148.1 ± 134.0
34.8
60.0
58.4 ± 22.6 4.8 ± 3.8
21.9 0.5
43.1 2.0
61.3 4.0
75.3 7.0
86.3 10.9
89.0 12.2
99.0 19.0
80.9 ± 18.9
50.2
71.5
86.0
96.9
99.0
99.6
99.9
6
H.A. Jeng et al.
than WHO reference levels (concentration > 15 × 106/mL, motility ≥ 40 %, and vitality ≥ 58 %) (WHO 2010). Age, drinking, and smoking were considered as the confounding factors. We then constructed a full multivariable linear regression model for each semen quality parameter as the dependent variable and individual ln-transformed As, Zn, Cd, Cu, and Pb as a predictor along with age (continuous variable), smoking (dichotomous), and drinking (dichotomous), BMI (continuous), and ln-transformed urinary creatinine (continuous) as covariates. Regression results are presented as the change in semen quality measure associated with a unit increase in ln-transformed metal concentration. Data analysis was performed using SPSS for Windows (Version 22).
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Results Table 1 summarizes demographic characteristics of the participants stratified by semen quality per WHO reference value categorizations. Among 196 participants, a total of 62 human subjects with semen parameters (concentration, motility, viability, and motility) met WHO reference values. All participants were Asian, with a mean age of 38 ± 10 years and BMI of 25 ± 4.1 kg/m2. Almost 50 % of participants did not smoke and drink. There was a difference in percentage of drinking between those with normal motility and those below normal motility. But none of the variances was statistically significant (p = 0.11). Table 2 describes the concentration of metals in seminal plasma and urine. Seminal plasma Pb and As concentrations were respectively in 79 and 51 % of samples below the LOD. Seminal plasma Cu and Se concentrations in all the samples were detectable, while Cd concentrations in 17 % of the samples were below the LOD. Urinary metals, e.g. Zn, Cd, Cu, and As, in most urine samples (> 98.5 %) were detectable, with 4.2 % of urinary Pb levels contained in 4.2 % of samples below the LOD. Tables 3 and 4 summarize comparison of metal concentrations in two categories of each semen parameter, based on the cut points suggested by the WHO to determine Table 3. Metals in seminal plasma classified by semen parameters according to the WHO reference levels. Cu Sperm concentration ≥ 15 × 106/ml 137.8 ± 210.1 < 15 × 106/ml 303.8 ± 258.8 p values 0.023 Motility ≥ 40 % 145.6 ± 307.5 < 40 % 146.4 ± 164.8 p values 0.976 Viability ≥ 58 % 160.6 ± 297.9 < 58 % 143.0 ± 183.0 p values 0.697 Normal morphology ≥4% 143.4 ± 182.1
International Journal of Environmental Health Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cije20
Role of low exposure to metals as male reproductive toxicants a
b
c
Hueiwang Anna Jeng , Yeou-Lih Huang , Chih-Hong Pan & Norou d
Diawara a
School of Community and Environmental Health, Old Dominion University, Norfolk, VA, USA b
Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan c
Occupational Safety and Health, Occupational Medicine, Taipei, Taiwan d
Mathematics & Statistics Department, Old Dominion University, Norfolk, VA, USA Published online: 01 Oct 2014.
To cite this article: Hueiwang Anna Jeng, Yeou-Lih Huang, Chih-Hong Pan & Norou Diawara (2014): Role of low exposure to metals as male reproductive toxicants, International Journal of Environmental Health Research, DOI: 10.1080/09603123.2014.958137 To link to this article: http://dx.doi.org/10.1080/09603123.2014.958137
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
International Journal of Environmental Health Research, 2014 http://dx.doi.org/10.1080/09603123.2014.958137
Role of low exposure to metals as male reproductive toxicants Hueiwang Anna Jenga*, Yeou-Lih Huangb, Chih-Hong Panc and Norou Diawarad a
School of Community and Environmental Health, Old Dominion University, Norfolk, VA, USA; Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan; cOccupational Safety and Health, Occupational Medicine, Taipei, Taiwan; dMathematics & Statistics Department, Old Dominion University, Norfolk, VA, USA
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b
(Received 31 March 2014; final version received 8 July 2014) The objective of the study was to examine the associations between environmentally relevant low metal concentrations and semen quality parameters in men. The concentrations of zinc (Zn), copper (Cu), cadmium (Cd), arsenic (As), selenium (Se), and lead (Pb) in the seminal plasma and urine were measured from 196 male human subjects in Taiwan. Urinary Cd concentrations were negatively associated with sperm viability (p = 0.006). Seminal plasma Cu concentrations of the normal group (≥ 15 × 106/ml) were significantly lower than those of the abnormal group (p = 0.023). However, the linear regression analysis showed a weak association between Cu concentration and sperm concentration, along with other semen parameters. No significant relationship between other metals (As, Pb, Zn, and Se) and semen quality was observed. Keywords: sperm concentration; viability; motility; morphology; metals
Introduction Due to the widespread presence of metals in the environment, the general population is exposed to low concentrations of metals through intake of contaminated food and water, contact with contaminated soil, or by breathing in dust or air (Meeker et al. 2008). Metals, such as lead (Pb), copper (Cu), zinc (Zn), and cadmium (Cd), have been reportedly linked with alteration of semen quality. Seminal plasma Pb of paint factory workers and the general population adversely affected sperm motility, morphology, and concentration (Eibensteiner et al. 2005; Naha & Manna 2007; Telisman et al. 2007; Meeker et al. 2008). Cd is also among the metals being found to associate with reduced sperm concentration (Telisman et al. 2000; Xu et al. 2003; Colagar & Marzony 2009), motility (Xu et al. 2003), and percentages of normal sperm morphology and viability (Xu et al. 2003; Colagar & Marzony 2009). Cu is necessary and useful for maintaining good reproductive health since it may act as a cofactor for a variety of important enzymes required for sperm development (Huang et al. 2000; Telisman et al. 2000). Despite its beneficial role, above certain levels, Cu proves to be harmful (Telisman et al. 2000). Similar to Cu, Zn could be beneficial for male reproductive health by acting as one of the factors responsible for antibacterial activity of the seminal plasma or possibly playing a role in sperm production, sperm viability, and degradation. Zn deficiency could lead to decreased testicular *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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weight, shrinkage of seminiferous tubules and gonadal dysfunction (Chia et al. 2000). There have been conflicting results about correlations of Zn concentrations in human seminal plasma and semen quality, including sperm concentration, motility, and viability (Dawson et al. 1998; Chia et al. 2000). To date, human data on some metals have been limited (e.g. As) or inconsistent across studies (e.g. Zn). Also, there is limited data on simultaneously examining semen quality associated with metals in different biological matrixes, such as urine and seminal plasma. Furthermore, there is limited data on the potential role of low, environmentally relevant exposure to metals in general populations in relation to male reproductive outcomes. This study aimed to examine the association between exposure to low concentrations of trace metals in seminal plasma and urine and semen quality (concentration, morphology, viability, and motility) of Taiwanese males in the general population. Semen quality serves as biological endpoints to assess the reproductive status of male individuals in this study and determine potential reproductive toxicity of metals. Materials and methods Human subjects A total of 281 human subjects were screened to determine eligibility for participation in this cross-sectional study. The screening occurred when they had their annual health examination at the Kaohsiung Municipal Hsiao-Kang Hospital, a main municipal hospital in the southern region of Taiwan. The criteria for selecting human subjects included males between 20 and 65 years old, no medical treatments affecting spermatogenesis (chemotherapy, radiotherapy, and vasectomy), no self-reported diabetes mellitus, cirrhosis or any other liver disease or neoplastic condition, no use of mood stabilizers, medications that affect the endocrine system, and no obesity class II and III according to the National Institutes of Health guidelines (NIH 2000). The status of fertility was not included as a criterion, since that reproductive status was not the focus of this study. However, we excluded those who had medical issues that prevented them from providing semen voluntarily. A total of 213 human subjects were found eligible and included in this study. The sample size was sufficient to yield a power of 85 % with a one-way analysis of variance at the 5 % level of significance. All participants were fully informed about the objective of this study and signed the consent form. The participants voluntarily provided semen and a spot of urine. They filled out a questionnaire describing demographic characteristics (age, height, weight, education, and marital status), smoking status, occupational exposure, and second-hand smoke exposure. Body mass index (BMI) for each human subject was calculated based on height and weight. The study was approved by the Institutional Review Boards at Old Dominion University and Kaohsiung Medical University. Semen quality analysis A semen sample was produced on-site by masturbation into a sterile plastic specimen cup. After collection, the sample was liquefied at 37 °C for 20 min before analysis. The participants were instructed to abstain from ejaculation for at least three days before semen collection. Each semen sample was collected by masturbation. All semen samples were analyzed for sperm concentration and motility according to World Health Organization guidelines (WHO 2010). Evaluation of percentage motility was done by manual
International Journal of Environmental Health Research
3
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assessment to quantify percentage of moving sperm compared with non-motile cells for at least 10 microscopic fields. For the morphology assessment, 300 sperm per sample were evaluated from air-dried Papanicolaou-stained preparations and classified as either normal or abnormal according to WHO’s strict criteria (WHO 2010). Results were expressed as percent normal sperm. Total sperm count was calculated as the product between sperm concentration and ejaculate volume. Total motile sperm was calculated as the product between total sperm count and progressive motility. All of the procedures were performed by the same laboratory technician. The mean inter-individual coefficients of variation were 18.2, 12.3, and 15.2 % for sperm concentration, motility, and viability, respectively Determination of metals in seminal plasma The semen samples were centrifuged at 1000 rpm for 5 min to separate sperm and seminal plasma. Then, 300 μL of each seminal plasma sample was transferred into a pre-cleaned 1.5 mL microtube and treated with 300 μL concentrated nitric acid at room temperature for 20 min. After homogenization, the mixture was diluted 5, 5, 1, 200, and 5-fold with deionized water for Cd, Cu, Pb, Zn, and As, respectively. After vigorous shaking, the sample solution was centrifuged at 12,000 rpm for 5 min. The obtained supernatant was directly analyzed by flame atomic absorption spectrometry (FAAS) for Zn, electrothermal atomic absorption spectrometry (ETAAS) for Cu, Cd, Se, and Pb, and hydride generation atomic absorption spectrometry (HGAAS) for As. The accuracy was validated by the standard spiked method, and the As, Cd, Cu, Pb, Se, and Zn concentrations in spiked semen samples were obtained by similar analytical methods. Recovery of the target analytes in spiked semen ranged from 95.10 to 105.97 %. The calibration curves were obtained after analyzing the standard series by FAAS, ETAAS, HGAAS as described, respectively. A good linearity was obtained at the concentration range of 5–30 (r2 = 0.9973), 0.5–2.5 (r2 = 0.9999), 5–50 (r2 = 0.9990), 5–60 (r2 = 0.9997), 1–6 (r2 = 0.9991), and 250–1000 (r2 = 0.9989) μg/L for As, Cd, Cu, Pb, Se, and Zn, respectively. Limits of detection (LOD) for As, Cd, Cu, Pb, Se, and Zn were 2.59, 0.03, 1.03, 0.98, 0.02, and 0.065 μg/L. Limits of quantitation (LOQ) for As, Cd, Cu, Pb, Se, and Zn were 8.46, 0.09, 2.95, 3.14, 0.07, and 0.217 μg/L, respectively. The concentrations of metals in seminal plasma were expressed as μg/L. Determination of metals in urine The urine samples were prepared as follows: 500 μL of urine was transferred into a precleaned 4 mL microtube and treated with aliquot of concentrated nitric acid at room temperature for 20 min. Then, the mixture solution was diluted 1, 1, 1, and 200-fold with deionized water for Cd, Cu, Pb, and Zn, respectively. After vigorous shaking, the urine samples were directly analyzed by FAAS for Zn, ETAAS for Cu, Cd, Pb, and Se. For As, urine samples were filtrated using a 0.45 μm hydrophilic polyethersulfone membrane prior to measurement by HGAAS. The accuracy was validated by determining the contents of As, Cd, Cu, Pb, Se, and Zn in standard reference material NIST-2670a human urine. The urine SRM 2670a was diluted similarly prior to analysis and the recoveries ranged from 93.05 to 108.17 %. The accuracy was also validated by the standard spiked method, with the As, Cd, Cu, Pb, Se, and Zn concentrations in spiked semen samples obtained by similar analytical methods. Recovery of the target analytes in spiked semen ranged from 93.05 to 104.91 %. The calibration curves were obtained at the
47.6 52.4 28.6 1.6 69.8 19.3 56.5 24.2
50.9 49.1
30.5 2.4 67.1
30.5 49.4 20.1
33.3 16.7 50.0
24.2 1.3 74.5
50.0 50.0
35.7 ± 11.6 24.2 ± 1.8
Concentration < 20 × 106/ml N=6
48.6 40.0 11.4
20.6 2.9 76.5
48.6 51.4
43.8 ± 9.4 25.6 ± 4.4
Motility < 40 % N = 31
61.9 23.8 14.3
23.8 4.8 71.4
50.0 50.0
46.1 ± 8.6 25.9 ± 4.1
Vitality < 58 % N = 21
45.5 39.4 15.2
36.9 3.1 60.0
60.6 39.4
38.7 ± 10.0 25.1 ± 4.4
Normal morphology < 4 % N = 66
*The comparison group was comprised of men with all four semen parameters higher than the reference levels according to the WHO reference levels.
36.8 ± 9.7 24.8 ± 3.9
Comparison group* N = 62
38.4 ± 9.9 25.0 ± 4.1
All
Demographic characteristics of the participants by semen quality parameters classified according to the WHO reference levels.
Age (years, mean ± SD) BMI (kg/m2, mean ± SD) Smoking status (%) Yes No Drinking (%) Yes Yes/Quit No Education (%) < High school High school College/Post college
Table 1.
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4 H.A. Jeng et al.
International Journal of Environmental Health Research
5
concentration range of 5–60 (r2 = 0.9976), 1–5 (r2 = 0.9998), 10–80 (r2 = 0.9991), 10–90 (r2 = 0.9994), 20–100 (r2 = 0.9991), and 100–800 (r2 = 0.9999) μg/L for As, Cd, Cu, Pb, Se, and Zn, respectively. The concentrations of As, Cd, Cu, Zn, Pb, and Se in urine samples were obtained by the general standard curve method. Concentrations of metals were adjusted by using urinary creatinine concentrations and expressed as ng/g creatinine. LOD for As, Cd, Cu, Pb, Se, and Zn were 2.59, 0.03, 1.03, 0.98, 0.02, and 0.065 μg/L, respectively. LOQ for As, Cd, Cu, Pb, Se, and Zn were 8.46, 0.09, 2.95, 3.14, 0.07, 0.217 μg/L, respectively. Urine volumes were adjusted by creatinine levels. The concentrations of metals were expressed as μg/g of creatinine.
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Statistical analysis Descriptive analysis was conducted to determine the mean distribution of demographics, semen quality parameters, and metal concentration in seminal plasma and urine. Mean concentration of each metal was calculated based on two separate groups of each semen parameter based on the WHO reference values. Mean metal concentrations in two categories of each semen parameter were compared using the Student’s t-test after ascertaining homogeneity of variance between the two groups. Association between exposure categories for each metal and sperm concentration, motility and vitality was assessed by binary logistic regression, where subjects were dichotomized in categories less than or greater Table 2. Distribution of semen quality and metals in seminal plasma, urine, and urine adjusted for creatinine levels. All Metal concentration Zn Cd Cu As Se Pb Metal concentration Zn Cd Cu As Se Pb Metal concentration Zn Cd Cu As Se Pb Semen quality Concentration (106/ml) Motility (% motile) Morphology (% normal) Viability (%)
10th
25th
50th
75th
in seminal plasma (μg/L) 169.3 ± 97.9 70.6 101.0 140.0 208.8 0.5 ± 0.3 0.38 0.4 0.6 1.4 170.8 ± 210.1 66.9 81.7 116.2 170.4 4.8 ± 5.5 1.3 1.3 1.3 7.3 249.1 ± 89.1 132.1 174.9 247.1 315.2 0.6 ± 1.1 0.5 0.5 0.5 0.5 in urine (μg/L) 602.0 ± 576.0 98.2 176.0 447.2 849.4 0.7 ± 0.6 0.2 0.4 0.9 1.4 14.8 ± 13.9 3.1 5.2 11.5 20.3 133.2 ± 146.4 12.7 33.3 77.1 177.6 20.4 ± 13.8 8.3 12.4 17.4 24.7 5.4 ± 6.4 0.3 0.5 3.1 8.5 in urine adjusted for creatinine (μg/g creatinine) 419.0 ±345.8 147.2 244.7 349.4 502.0 0.5 ± 0.4 0.1 0.2 0.4 0.8 12.3 ± 11.6 3.8 6.0 10.1 13.8 91.1 ± 91.9 19.4 37.4 64.1 110.8 15.4 ± 10.8 5.7 7.5 12.6 20.3 4.4 ± 7.8 0.2 0.8 2.5 6.1 111.5 190.0
90th
95th
Maximum
304.6 1.5 252.2 12.7 353.3 0.5
371.2 1.5 440.1 16.3 362.5 0.5
612.0 1.5 1756.8 32.8 609.7 12.6
1414.3 1669.3 2.4 5.8 31.8 36.1 336.1 433.8 35.6 45.7 15.1 17.8
3279.6 2.9 94.1 754.5 84.3 34.7
707.8 1.1 22.1 184.5 31.0 8.5
819.7 1.4 32.5 297.7 39.3 12.1
2938 2 88 686 58 72
273.0
313.5
905.0
148.1 ± 134.0
34.8
60.0
58.4 ± 22.6 4.8 ± 3.8
21.9 0.5
43.1 2.0
61.3 4.0
75.3 7.0
86.3 10.9
89.0 12.2
99.0 19.0
80.9 ± 18.9
50.2
71.5
86.0
96.9
99.0
99.6
99.9
6
H.A. Jeng et al.
than WHO reference levels (concentration > 15 × 106/mL, motility ≥ 40 %, and vitality ≥ 58 %) (WHO 2010). Age, drinking, and smoking were considered as the confounding factors. We then constructed a full multivariable linear regression model for each semen quality parameter as the dependent variable and individual ln-transformed As, Zn, Cd, Cu, and Pb as a predictor along with age (continuous variable), smoking (dichotomous), and drinking (dichotomous), BMI (continuous), and ln-transformed urinary creatinine (continuous) as covariates. Regression results are presented as the change in semen quality measure associated with a unit increase in ln-transformed metal concentration. Data analysis was performed using SPSS for Windows (Version 22).
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Results Table 1 summarizes demographic characteristics of the participants stratified by semen quality per WHO reference value categorizations. Among 196 participants, a total of 62 human subjects with semen parameters (concentration, motility, viability, and motility) met WHO reference values. All participants were Asian, with a mean age of 38 ± 10 years and BMI of 25 ± 4.1 kg/m2. Almost 50 % of participants did not smoke and drink. There was a difference in percentage of drinking between those with normal motility and those below normal motility. But none of the variances was statistically significant (p = 0.11). Table 2 describes the concentration of metals in seminal plasma and urine. Seminal plasma Pb and As concentrations were respectively in 79 and 51 % of samples below the LOD. Seminal plasma Cu and Se concentrations in all the samples were detectable, while Cd concentrations in 17 % of the samples were below the LOD. Urinary metals, e.g. Zn, Cd, Cu, and As, in most urine samples (> 98.5 %) were detectable, with 4.2 % of urinary Pb levels contained in 4.2 % of samples below the LOD. Tables 3 and 4 summarize comparison of metal concentrations in two categories of each semen parameter, based on the cut points suggested by the WHO to determine Table 3. Metals in seminal plasma classified by semen parameters according to the WHO reference levels. Cu Sperm concentration ≥ 15 × 106/ml 137.8 ± 210.1 < 15 × 106/ml 303.8 ± 258.8 p values 0.023 Motility ≥ 40 % 145.6 ± 307.5 < 40 % 146.4 ± 164.8 p values 0.976 Viability ≥ 58 % 160.6 ± 297.9 < 58 % 143.0 ± 183.0 p values 0.697 Normal morphology ≥4% 143.4 ± 182.1
